Follicular waves during the estrous cycle

Follicular wave emergence in cattle is characterized by the sudden (within 2–3 days) growth of 8–41 small follicles that are initially detected by ultrasonography at a diameter of 3–4 mm (Figures 24.6 and 24.7).^{20,25,26,31,32} The growth rate is similar among follicles of the wave for about 2 days, after which one follicle is then selected to continue growth (dominant follicle) while the remainder become atretic and regress (subordinate follicles). In both two- and three-wave estrous cycles, emergence of the first follicular wave occurs consistently on the day of ovulation (day 0). Emergence of the second wave occurs on day 9 or 10 for two-wave cycles, and on day 8 or 9 for three-wave cycles. In three-wave cycles, a third wave emerges on day 15 or 16. Under the influence of progesterone (e.g., diestrus, pregnancy), dominant follicles of successive waves are anovulatory and undergo atresia.³³ The dominant follicle present at the onset of luteolysis becomes the ovulatory follicle, and emergence of the next wave is delayed until the day of the ensuing ovulation. The corpus luteum begins to regress earlier in two-wave cycles (day 16) than in three-wave cycles (day 19), resulting in a correspondingly shorter estrous cycle (20 days vs. 23 days, respectively). Hence, the proverbial 21-day estrous cycle of cattle exists only as an average between two- and three-wave cycles.

Two-wave versus three-wave patterns

The majority of bovine estrous cycles (>95%) are composed of either two or three follicular waves (Figure 24.7).^34,35 Some have reported a preponderance (>80%) of either the two-wave or the three-wave pattern during an interovulatory interval, while others have reported a more even distribution. There appears to be no breed- or age-specific predilection for a given wave pattern in Bos taurus. However, an increase in the proportion of three-wave patterns has been associated with a low plane of nutrition^36,37 and heat stress.^38,39 In Bos indicus, no seasonal effect on wave pattern was detected,⁴⁰ but the pattern was influenced by parity. The majority of Nelore heifers (65%) exhibited a three-wave pattern, whereas the majority of cows (83%) exhibited a two-wave pattern.⁴¹ Others have reported that up to 27% of estrous cycles in Bos indicus cows consist of four waves of follicular development, compared with only 7% in Bos indicus heifers (reviewed in Ref. 42).

Predictive factors associated with a two- versus three-wave pattern may provide insight into mechanisms controlling the pattern, and have important implications for breeding management and the development of effective protocols for ovarian synchronization, superstimulation, and fixed-time artificial insemination. Pregnancy rates in cattle with two- versus three-wave patterns were compared^43–45 based on the notion that the preovulatory follicle in the two-wave pattern grows for a longer period³¹ and may therefore contain a relatively aged oocyte. However, results have been contradictory; pregnancy rates did not differ between two- versus three-wave cycles in some studies,^43,45 whereas a lower pregnancy rate following ovulation at the end of the two-wave pattern was reported in another study.⁴⁴

In a more recent study involving ultrasonographic data from 91 interovulatory intervals,³⁵ two- and three-wave patterns of follicular development were compared to determine the repeatability and predictive characteristics of a given wave pattern. Two-wave cycles were 3 days shorter than three-wave cycles (19.8 ± 0.2 vs. 22.5 ± 0.3; P < 0.01). The majority of cycles of 21 days or less were of the two-wave pattern (88%; P < 0.05), while the majority of cycles 22 days or longer were of the three-wave pattern (78%; P < 0.05). The proportion of successive cycles in which the pattern remained the same (i.e., repeatability) was more than twofold greater than the proportion of cycles that changed patterns (70% vs. 30%; P < 0.01). The repeatability of wave pattern, and the proportion of two- versus three-wave patterns within the herd were not affected by the season of the year; in other words not affected by pasture-based (spring and summer) versus non-pasture-based (fall and winter) seasons. Interestingly, the strongest correlate to the number of waves in the interovulatory interval was the duration of follicular dominance of Wave 1. The duration of dominance of the Wave 1 dominant follicle was 3 days longer, and the onset of regression was later in two-wave patterns than in three-wave patterns (P < 0.01). Dominance of Wave 1 was associated with a subsequent delay in the attainment of maximum diameter by the dominant follicle of Wave 2, as well as early onset of luteolysis (Figure 24.7). Results supported the hypothesis that factors which influence the development of the dominant follicle of Wave 1 are responsible for regulating the wave pattern.

Two ovaries, one wave

The two ovaries act as a single unit – one ovary does not have follicular waves independent of the other. The ovaries respond in unison to factors regulating follicular wave dynamics, hence a follicular wave is the result of multiple follicle growth in both ovaries (Figures 24.6 and 24.7). In a critical study of the intraovarian relationships among follicles and the corpus luteum, Ginther et al.⁴⁶ concluded that the dominant follicle suppresses subordinates and new wave emergence via systemic rather than local channels. The presence of a dominant follicle in one ovary had no effect that could not be seen equally in the other ovary. Hence, no intraovarian relationships were found in the location of successive dominant follicles during the estrous cycle; in other words, the side of anovulatory and ovulatory dominant follicles was random. These observations are consistent with the findings of early studies on the effects of unilateral ovariectomy. Removal of the ovary with the largest follicle resulted in an increase in follicle development in the remaining ovary, but removal of the ovary with’out the largest follicle resulted in no such compensatory effect (cited in Ref. 46). While intrafollicular (autocrine and paracrine) factors are important for growth, health, and demise of an individual follicle, there is no convincing in vivo documentation of one follicle affecting the health/regression status of its neighbors directly.

Asymmetry in follicle dynamics in the left and right ovaries has been used to elucidate local versus systemic mechanisms of control of ovarian function, and some have reported greater follicular activity and proportion of ovulations in the right ovary in cattle (~60%),^12,22 whereas others report no such differences.^26,31 Ovarian follicular asymmetry has also been attributed to the presence of a corpus luteum. A positive intraovarian effect of the corpus luteum on the development of small antral follicles (≤3 mm) has been documented in sheep⁴⁷ and cattle,²¹ but the positive effect did not extend to large dominant follicles.^21,46 Conversely, the corpus luteum of pregnancy has been associated with a negative intraovarian effect on the dominant follicle.^46,48 Dominant follicles of successive waves were more frequently (75–80%) found in the ovary contralateral to the corpus luteum during pregnancy. The cause of the negative local association between the corpus luteum and the follicles is unknown but it may be directly related to the conceptus rather than to the corpus luteum. Support for this notion is found in the observation that there were no ovarian asymmetries during the first two follicular waves in pregnant cattle or in 10 successive follicular waves in unmated progesterone-treated heifers.³³ The latter observation, however, is confounded by the fact that exogenous progesterone did not forestall luteolysis in treated heifers; luteal regression occurred at the expected time for a nonpregnant animal regardless of progesterone treatment. However, an experimental design involving hysterectomy during mid-diestrus permitted direct comparison of the effects of a persistent corpus luteum due to hysterectomy or pregnancy.⁴⁹ Results demonstrated that unilateral attenuation of follicle development was due to the conceptus or the ipsilateral gravid uterine horn, and not the corpus luteum.

Hormonal interplay controlling follicular wave dynamics

Results of early studies of follicle dynamics gave rise to the hypothesis that the dominant follicle suppresses the growth of the subordinates in the existing wave, and suppresses the emergence of the next follicular wave.^31,32 Support for this hypothesis was provided in a series of studies involving systemic treatment with the proteinaceous fraction of follicular fluid and by electrocautery of the dominant follicle.^50–52 The applied implications of these findings were immediate and far-reaching, and marked a new era for ovarian synchronization and superstimulation in cattle.^53–55

Emergence of a follicular wave and selection of the dominant follicle are temporally and causally associated with a rise and fall in circulating concentrations of follicle-stimulating hormone (FSH)⁵¹ (Figure 24.8). Emergence of a follicular wave is preceded by a surge in plasma FSH concentrations in both spontaneous waves and induced waves.^51,56 Surges in FSH preceding each wave are of similar magnitude: mid-cycle surges are similar in amplitude and breadth as those of the preovulatory gonadotrophin surge.^51,56 Follicular products, especially those from the dominant follicle, are responsible for suppressing FSH release and therefore the emergence of the next follicular wave⁵⁷ (Figure 24.9). A direct relationship between the number of follicles emerging in response to the FSH surge and the subsequent degree of FSH suppression^58–60 documents that all follicles of the new wave contribute to FSH suppression.

Estradiol and inhibin-A and -B are the principal follicular products responsible for suppressing FSH.^57,61–65 Inhibin-A is produced by all the small growing follicles of the wave and appears to be the most important suppressor of FSH during the first 2 days of wave emergence; estradiol secreted from the dominant follicle is the most important FSH suppressor thereafter.^66–69 The nadir in FSH is reached 4 days after wave emergence and levels remain low for next 2–3 days. At the end of the period of dominance (i.e., at ovulation, or the mid-static phase of an anovulatory dominant follicle), circulating concentrations of FSH begin to rise over the next 2 days and peak about 12–24 hours before emergence of the next follicular wave, when the future dominant follicle is 4–5 mm in diameter. If an existing dominant follicle is removed (i.e., follicular ablation), a surge in FSH begins within the next 12 hours and results in emergence of a new follicular wave within the next 24 hours.^52,70 Interestingly, FSH only rises again when inhibin-A concentrations reach nadir, despite the fact that estradiol declines 2 days earlier.⁶³

An inverse relationship between circulating progesterone concentrations and dominant follicle growth was suggested by the observations that (i) in three-wave estrous cycles, the maximum diameter of the dominant anovulatory follicle was greater for Wave 1 (under minimal luteal dominance during its growing phase) than for Wave 2 (under maximum luteal dominance); (ii) the maximum diameter attained by the dominant anovulatory follicle was greater for Wave 1 than for all subsequent waves in pregnant and nonmated progesterone-treated heifers; and (iii) high concentrations of progesterone (endogenous plus exogenous) resulted in a smaller maximum diameter of the dominant follicle of Wave 2 compared with subsequent waves (exogenous progesterone only) in nonmated progesterone-treated heifers (reviewed in Ref. 71). A cause-and-effect relationship was established in studies in which progesterone was shown to suppress the dominant follicle during its growing phase in a dose-dependent manner⁷¹ through the negative feedback effect of progesterone on luteinizing hormone (LH) pulsatility.^72–74 The suppressive effects on the dominant follicle were not mediated by decreased circulating FSH since progesterone treatment did not suppress FSH.⁷¹ High plasma progesterone during follicular growth resulted in a smaller shorter-lived dominant follicle, an earlier surge in FSH preceding the next follicular wave, and hastened emergence of the next follicular wave (Figure 24.10). Conversely, low concentrations of progesterone resulted in an oversized persistent dominant follicle, a delayed surge in FSH, and delayed emergence of the next wave.⁷¹ The oversized dominant follicle in the low-dose situation would have been considered a pathological follicular cyst because of its size and persistence for more than 10 days in the absence of a corpus luteum.^75,76 Low-level progesterone exposure (and therefore less inhibition of LH), at the time the dominant follicle normally stops growing, appears to be a fundamental component in the etiology of ovarian follicular cysts.⁷¹

Selection of the dominant follicle

In monovular species, the term “selection” refers to a process by which a single follicle becomes functionally and morphologically dominant over other follicles of a wave. In cattle, the mechanism of selection of the dominant follicle is based on relative reliance of follicles within a wave on FSH, and their relative responsiveness to LH. The transient rise in FSH permits sufficient follicular growth so that some (not all) follicles acquire LH responsiveness. The ability to respond to LH imbues the follicle with the ability to survive without FSH.

It remains unclear precisely when the process begins, but selection of the dominant follicle is associated with declining levels of FSH in circulation during the first 3 days of the wave.^51,77 Small follicles of the emerging wave (<6 mm) are dependent on elevated circulating concentrations of FSH for continued development. In the face of the post-surge decline in FSH, the growth of most of the follicles of a wave stops and they begin to regress within 2–5 days of emergence. Conversely, the follicle destined to become dominant can maintain cell proliferation and estradiol production despite declining FSH concentrations, an ability that appears to be brought about by maintaining high FSH receptor mRNA expression and FSH-binding affinity.^28,29,78,79

Divergence in the growth profiles (defined as a significant difference in diameter) of the largest and second largest follicle is a manifestation of the selection process and is apparent by 2.5 days after wave emergence (Figures 24.8 and 24.9).⁷⁷ Similarly, follicle “deviation” (defined as a difference in growth rate between the dominant and subordinate follicles) is manifest at 2.5 days after wave emergence, when the future dominant follicle reaches a mean diameter of 8.5 mm in Holstein heifers (reviewed in Ref. 80). Continued development of the dominant follicle, beyond the 8-mm threshold (point at which divergence in the growth rate of the largest two follicles becomes evident), is associated with a transition from FSH to LH dependence.^66,68,81 Granulosa cells from the dominant follicle acquire an enhanced ability to bind LH compared with that of the subordinates, and have greater LH receptor mRNA expression.^{29,78,79,82,83} Although a slight increase in LH receptor mRNA expression was detected in granulosa cells from the largest follicle a few hours before the onset of deviation,⁸⁴ it is not yet clear whether the observed increase in LH receptor expression in the dominant follicle is the cause of dominance or a consequence of the selection process. Perhaps the increased LH receptor expression is merely a reflection of a follicle with a developmental advantage before such an advantage becomes ultrasonographically apparent. In this regard, results of a critical study of the development of small follicles support the hypothesis that the follicle destined to become dominant has a size advantage over the others in the wave from its earliest detection at 1 mm (Figure 24.11) (see section Small antral follicles).